Despite escalating efforts to identify antiprostate cancer agents, advanced prostate cancer is still a universally progressive and fatal disease. This unusual resistance to conventional chemotherapeutic agents has led to the exploration of a variety of novel therapeutic strategies, including suicide gene therapy (Rodriguez, R. and Simons, J. W. (1999) Urology 54:401-406). This strategy relies on the delivery to cancer cells of genes that are either directly toxic or that produce toxic metabolites when administered with a prodrug. Examples of such a strategy include herpes simplex thymidine kinase, which can phosphorylate ganciclovir (an acyclic nucleoside analogue of 2′-deoxyguanosine), into the toxic monophosphate form. Other examples include cytosine deaminase and purine nucleoside phosphorylase. Because these suicide genes poison DNA synthesis by promoting abortive DNA chain elongation, they are uniquely effective at targeting rapidly dividing cells (e.g., leukemias, lymphomas, certain childhood malignancies, and germ cell tumors). Unfortunately, because <3% of prostate cancer cells are actively dividing at any given moment, this strategy is conceptually less appealing in the context of prostate cancer (Berges, R. R. et al. (1995) Clin. Cancer. Res. 1:473-480). Thus, there is a need in the art for the development of suicide gene therapeutic agents that are active against quiescent cells (i.e., cell cycle independent), yet potent and regulated.
Diphtheria toxin (DT) is a potent cellular toxin that poisons protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (Greenfield, L. et al. (1983) Proc. Natl. Acad. Sci. USA (1983) 6853-6857) and kills cells primarily by an apoptosis-mediated pathway (Kochi, S. K. and Collier, R. J. (1993) Exp. Cell Res. 208:296-302). In certain situations (e.g., mutant p53 expression) the cells may die by necrosis rather than apoptosis; however, the pharmacokinetics is similar regardless of the pathway of cell death (Rodriguez, R. et al. (1998) Prostate 34:259-269). DT is composed of three functional domains located in two subunits, the A chain and B chain, which are joined by a disulfide bond (Bennet, M. J. and Eisenber, D. (1994) Protein Sci. 3:1464-1475). The A chain of DT has the catalytic domain, whereas the B chain comprises the receptor binding and translocation domains. It has been estimated that a single molecule of DT is capable of killing a cell (Yamaizumi, M. et al. (1978) Cell 15:245-250); therefore, strategies must be used that limit its delivery to or expression in specific target cells. These strategies have included delivering expression constructs directly to diseased cells by liposomal gene transfer under the control of a regulatory element or tissue-specific promoter (Vingerhoeds, M. H. et al. (1996) FEBS Lett. 395:245-250; Konopka, K. et al. (1997) Biochim. Biophys. Acta 1356:185-197; Duzgunes, N. et al. (1999) Mol. Membr. Biol. 16:111-118; Kunitomi, M. et al. (2000) Jpn. J. Cancer Res. 91:343-350), or delivering the toxin A-chain molecules by virtue of fusion to cloned antibody fragments (Kreitman, R. J. (1999) Curr. Opin. Immunol. 11:570-578) or peptide ligands for cell-specific receptor-mediated endocytosis (Kelley, V. E. et al. (1988) Proc. Natl. Acad. Sci. USA 85:3980-3984; Arora, N. et al. (1999) Cancer Res. 59:183-188; Hall, P. D. et al. (1999) Leukemia (Baltimore) 13:629-633).
Previous attempts at limiting toxicity of DT-A by use of a tissue-specific promoter have led to variable results. For example, Maxwell et al. (Maxwell, I. H. et al. (1986) Cancer REs. 46:4660-4664) used a truncated form of the metallothionein promoter to demonstrate that basal expression of this promoter, even in the absence of heavy metals, resulted in substantial inhibition of protein synthesis. This inhibition could be augmented by the addition of an immunoglobulin enhancer element but only minimally by cadmium. The authors were never able to demonstrate true specific cytotoxicity but rather only a preferential cell susceptibility to DT-A-mediated cell death, presumably as a result of basal expression of this highly toxic gene. As a result, subsequent efforts by this group and others concentrated on introducing an attenuated mutant of DT-A (Maxwell, F. et al. (1987) Mol. Cell. Biol. 7:1576-1579) or on tightly regulating gene expression using prokaryotic control elements (Robinson, D. F. and Maxwell, I. H. (1995) Hum. Gene Ther. 6:137-143; Paulus, W. et al. (1997) J. Neurosurg. 87:89-95). In both cases, although preferential cell killing could be demonstrated, complete abolition of nonspecific cell killing was not achieved. As a follow-up study, Keyvani et al. (Keyvani, K. et al. (1999) Life Sci. 64:1719-1724) used the same tet repressor-based system as they had reported previously with the wild-type DT-A gene with an attenuated DT-A mutant and were still unable to demonstrate complete abolition of background expression and subsequent cell death.
There currently exists a need for additional methods for producing suicide gene therapy vectors for use in killing cancer cells that are specific and reliable. The present invention fulfills that need.